Transmembrane nfat inhibitory peptide

ABSTRACT

The present invention aims to solve the conventional problems by providing a peptide compound, in which when administered to a patient of immunologic disease, cardiac hypertrophy or a disease caused by NFAT activation, the period from administration to actual exhibition of effectiveness is short and there are no side effects or antigenicity. Specifically, the present invention relates to a membrane permeable NFAT inhibitory peptide comprising several consecutive arginines and a NFAT-activity inhibitory peptide sequence; a NFAT activation inhibitory agent comprising the peptide; and a immunosuppressive agent and cardiac hypertrophy suppressive agent, comprising the peptide compound as the active ingredient.

TECHNICAL FIELD

The present invention relates to a novel peptide compound comprising asignal sequence for biomembrane permeation. Furthermore, the presentinvention relates to a NFAT activation inhibitory peptide, animmunosuppressive agent and a cardiac hypertrophy suppressive agentcomprising several residues of arginine and VIVIT.

BACKGROUND ART

Conventionally, various compounds have been applied as animmunosuppressive agent to immunologic diseases such as rejection aftertransplant surgery and atopic dermatitis. For example, Cyclosporine A(CysA) and FK 506 are commonly known immunosuppressive agents. However,when administered to animals, these immunosuppressive agents are alsoknown to cause side effects such as loss of renal function,hypertension, decrease of insulin secretion amount and neurotoxicity.Immunosuppressive agents other than CysA and FK 506 have been also beenfound to cause various side effects and an immunosuppressive agenthaving fewer side effects is desired.

Currently, besides treatment by the above synthetic compounds, genetherapy, wherein a vector comprising a foreign gene is administered to ahuman, is attracting attention and studies at a clinical level are beingconducted for various diseases. However, the vector used for genetherapy has many problems that need to be solved, such as efficiency ofintroduction into cells, time required from administration to expressionof the protein and side effects when the vector is administered to ahuman. Also, various peptide preparations have been considered but atpresent, none are used clinically.

Cardiac hypertrophy refers to the condition of the heart becoming largerthan usual due to genetic background or pressure load and may lead toheart failure. However, a cardiac hypertrophy suppressive agent thatprevents progression of or promotes involution of cardiac hypertrophy isnot commercially available at present. This is because the currentlyknown cardiac hypertrophy suppressive agent has strong side effects andtherefore cannot actually be applied to a human. Consequently,medication that prevents or remedies cardiac hypertrophy and does nothave problems such as side effects is strongly desired.

DISCLOSURE OF INVENTION

The object of the present invention is to solve the above conventionalproblems and to provide a peptide compound, in which the time fromadministration to actually taking effect is short and which does nothave side effects or antigenicity. Particularly, the present inventionaims to provide a therapeutic agent for immunologic diseases and cardiachypertrophy. The present invention also relates to a NFAT activationinhibitory agent.

Japanese Patent Application No. 2000-358442 discloses that a peptidecomprising 9 to 13 residues of arginine, particularly 11 residues ofarginine, is extremely useful as signal sequence for biomembranepermeation. As a result of further studies, it has been found for thefirst time that an excellent immunosuppressive effect can be obtainedwhen a peptide compound obtained by fusion of 9 to 13 residues ofarginine and a peptide that inhibits the activity of Nuclear FactorActivated T cell (NFAT) is used in vivo. Furthermore, when this peptideis administered to a rat suffering from cardiac hypertrophy, thesymptoms of cardiac hypertrophy were found to improve considerably andthe present invention was achieved.

That is, the present invention relates to a peptide compound comprising9 to 13 residues of arginine and the amino acid sequence of SEQ ID NO.1.

The present invention also relates to a NFAT activation inhibitory agentcomprising a peptide compound comprising 9 to 13 residues of arginineand the amino acid sequence of SEQ ID NO. 1.

The present invention also relates to an immunosuppressive agentcomprising a peptide compound comprising 9 to 13 residues of arginineand the amino acid sequence of SEQ ID NO. 1 as an active ingredient.

The present invention also relates to a cardiac hypertrophy suppressiveagent comprising a peptide compound comprising 9 to 13 residues ofarginine and the amino acid sequence of SEQ ID NO. 1 as an activeingredient.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is a graph showing the transcription amount of IL-2 gene in cellscultured by adding 11R-VIVIT, FK 506 and 11R-VEET in the respectiveconcentrations. The vertical line represents the relative RNA amount ofIL-2 gene to GAPDH.

FIG. 2 is a graph showing the transcription amount of IL-2 gene inJurkat cells cultured for 60 hours in only a RPMI medium containing 10%fetal bovine serum, Jurkat cells cultured for 13 hours, 18 hours, 24hours, 36 hours and 60 hours after adding 11R-VIVIT and Jurkat cellscultured for 60 hours after adding FK 506. The vertical line representsthe relative RNA amount of IL-2 gene to GAPDH.

FIG. 3 is a graph showing the radioactivity of [³H]TdR incorporated intocells in a mixed lymphocyte test.

FIG. 4 is a graph showing the radioactivity of [³H]TdR incorporated intocells in a mixed lymphocyte test using cells obtained from a mouse towhich 11R-VIVIT, FK 506 and 11R-VEET are administered.

FIG. 5 is a graph evaluating the survival rate of transplanted betacells by Kaplan-Meier.

FIG. 6 is a microscope image showing the results of immunostainingtransplanted beta cells. 1 shows beta cells of a BALB/c mouse. 2 showsthe kidney of a diabetic model mouse.

FIG. 7 is a graph showing the insulin secretion amount of cellssubjected to the respective treatments by a relative value to control.FIG. 7(a) is a graph showing the insulin secretion amount of cellstreated with 11R-VIVIT of the respective concentrations by a relativevalue to control. FIG. 7(b) is a graph showing the insulin secretionamount of cells treated with FK 506 of the respective concentrations bya relative value to control. FIG. 7(c) is a graph showing the insulinsecretion amount of cells treated with 11R-VEET of the respectiveconcentrations by a relative value to control.

FIG. 8 is a graph showing the proliferation of cells subjected to therespective treatments by a relative value to control. FIG. 8(a) is agraph showing the proliferation of cells treated with 11R-VIVIT of therespective concentrations by a relative value to control. FIG. 8(b) is agraph showing the proliferation of cells treated with FK 506 of therespective concentrations by a relative value to control. FIG. 8(c) is agraph showing the proliferation of cells treated with 11R-VEET of therespective concentrations by a relative value to control.

FIG. 9 is a graph showing the results of analyzing the interventricularseptum wall thickness and the left ventricle anterior wall thickness ofa rat by ultrasonic echo. FIG. 9(a) is a graph showing theinterventricular septum wall thickness of control, AoB, CysAadministered AoB and IIR-VIVIT administered AoB. FIG. 9(b) is a graphshowing the left ventricle anterior wall thickness of control, AoB, CysAadministered AoB and IIR-VIVIT administered AoB.

FIG. 10 is a microscope image of a tissue specimen of a rat heart. FIG.10(a) is a microscope image showing a section of a normal rat heart.FIG. 10(b) is a microscope image showing a section of an AoB heart. FIG.10(c) is a microscope image showing a section of a CysA administered AoBheart. FIG. 10(d) is a microscope image showing a section of an11R-VIVIT administered AoB heart. 3 shows the left ventricle and 4 showsthe right ventricle.

FIG. 11 is a graph showing the blood test results of a rat. FIG. 11(a)is a graph showing ANP of control, AoB, CysA administered AoB andIIR-VIVIT administered AoB. FIG. 11(b) is a graph showing BNP ofcontrol, AoB, CysA administered AoB and IIR-VIVIT administered AoB.

BEST MODE FOR CARRING OUT THE INVENTION

Hereinafter, the peptide compound of the present invention and usesthereof are described in detail.

The peptide compound of the present invention comprises an amino acidsequence having several consecutive arginines and the amino acidsequence of SEQ ID NO. 1.

The number of consecutive arginine residues is preferably 9 to 13, mostpreferably 11. When the number of consecutive arginine residues is 8 orless or 14 or more, the efficiency of introducing the peptide compoundof the present invention into cells tends to become poor.

The peptide compound of the present invention also comprises a NFATactivity inhibitory peptide VIVIT. NFAT is a transcriptional regulatoryfactor that is activated when dephosphorylated by calcineurin and formsa family including NFAT 1, NFAT 2, NFAT 3 and NFAT 4. VIVIT is a peptideof the amino acid sequence represented by MAGPHPVIVITGPHEE (SEQ ID NO.1). VIVIT selectively inhibits the interaction between proteins thatbelong to the NFAT family and calcineurin, without influencing thephosphatase activity of calcineurin (Aramburu, J. et al., Science, Vol.285, pp. 2129 to 2133, 1999). In the amino acid sequence of SEQ ID NO.1, any amino acid residue can be substituted with a biologicallyequivalent amino acid residue as long as the amino acid sequenceinhibits NFAT and several amino acid residues can be added or deleted aslong as the amino acid sequence has biological activity equivalent toVIVIT.

Also, in addition to the above amino sequence comprising severalconsecutive arginines and the amino acid sequence of SEQ ID NO. 1, thepeptide compound of the present invention can have several amino acidresidues added, deleted or substituted, as long as the peptide compoundhas the same biological activity.

Furthermore, the peptide compound of the present invention can contain amarker for confirming introduction into cells. The marker is notparticularly limited and for example, fluorescein isothiocyanate(hereinafter referred to as FITC), GFP and rhodamine can be used.

The peptide compound of the present invention can be prepared by theusual artificial synthesis method or by an artificial synthesis machinethat is generally commercially available. Also, the peptide compound ofthe present invention can be prepared using a genetic engineeringmethod. For example, a recombinant vector, in which DNA encoding thepeptide sequence comprising a sequence of several consecutive argininesand a sequence of VIVIT is inserted, is prepared and after introducingthe recombinant vector in a suitable host cell, the host cell iscultured. Subsequently, by recovering the cultured substance, thepeptide compound of the present invention is obtained. After preparingthe peptide compound of the present invention, the obtained peptidecompound can be purified by a known method. The preparation andpurification methods of the peptide compound are common methods in thepresent field and can be easily conducted by one skilled in the art.

The peptide compound of the present invention can be used as asuppressive agent of NFAT activation. A suppressive agent of NFATactivation can be used as a therapeutic agent for diseases caused byNFAT activation. Examples of diseases caused by NFAT activation are forallergic diseases and cardiac hypertrophy.

The peptide compound of the present invention has the function ofinducing immunosuppression and can be used as an immunosuppressive agentfor transplant. The immunosuppressive agent of the present invention canalso be used as a therapeutic agent for immunologic diseases. Specificexamples of immunologic disease are rejection after transplant surgeryand atopic dermatitis.

The immunosuppressive agent of the present invention can be administeredparenterally, for example, intravenously, hypodermically,intramuscularly, transdermally and intrarectally. Particularly, becausedecomposition of the peptide compound can be prevented by introducingthe peptide compound directly into bloodstream, intravenous or subdermaladministration is preferable.

The form of the immunosuppressive agent of the present invention can bedetermined according to the administration method. Specifically,examples are liquid formulations, such as an aqueous solution and anemulsion, and ointment.

The effective amount of the immunosuppressive agent of the presentinvention can be determined accordingly in consideration of theadministration method and age, weight and medical conditions of thepatient and is usually 1 to 10 mg/kg converted to the active ingredient.However, the amount of the peptide compound of the present invention,which is the active ingredient, can be changed and is not limited to theabove range.

The immunosuppressive agent of the present invention is not particularlylimited as long as it comprises the peptide compound of the presentinvention as the active ingredient and other components that are usuallyused in this technical field can be added, such as a suitablepharmaceutical excipient, a suitable carrier, a solvent, a gel formingagent, an antioxidant, a diluent, an isotonizing agent and a pHstabilizer. These additives can usually be selected accordingly by oneskilled in the art.

The peptide compound of the present invention is effective as a cardiachypertrophy suppressive agent. The cardiac hypertrophy suppressive agentof the present invention can be administered parenterally, for exampleintravenously, hypodermically, intramuscularly, transdermally orintrarectally. Particularly, because introducing the peptide compounddirectly into bloodstream is important for of preventing decompositionof the peptide compound, intravenous administration is preferable.

The form of the cardiac hypertrophy suppressive agent of the presentinvention can be determined according to the administration method.Specifically, examples are liquid formulations, such as an aqueoussolution or an emulsion, and ointment.

The dosage of the cardiac hypertrophy suppressant of the presentinvention can be determined accordingly in consideration of theadministration method and age, weight and medical conditions of thepatient and is usually 0.1 to 2 mg/kg converted to the activeingredient. However, the amount of the peptide compound of the presentinvention, which is the active ingredient, can be changed and is notlimited to the above range.

The cardiac hypertrophy suppressive agent of the present invention isnot particularly limited as long as it contains the peptide compound ofthe present invention as the active ingredient and other components thatare usually used in this technical field can be added, such as asuitable pharmaceutical excipient, a suitable carrier, a solvent, a gelforming agent, an antioxidant, a diluent, an isotonizing agent and a pHstabilizer. These additives can usually be selected accordingly by oneskilled in the art.

Hereinafter, the peptide compound of the present invention is describedin more detail by means of Examples, but the present invention is notlimited thereto as long as the purpose and scope of the presentinvention are not deviated from.

EXAMPLE 1

A peptide compound comprising 11 consecutive arginine residues, VIVITand FITC was artificially synthesized (preparation commissioned to SigmaGenosis Japan, Inc.) and the peptide was purified by reversed-phaseHPLC. Hereinafter, a peptide comprising 11 consecutive arginine residuesand VIVIT to which 3 glycine residues are added is referred to as11R-VIVIT. The peptide sequence of 11R-VIVIT is shown in SEQ ID NO. 2.FITC is added to the amino terminal of 11R-VIVIT.

EXAMPLE 2

A six-week old C3H/HeN mouse H-2k (available from CLEA Japan, Inc.) wassubjected to intraperitoneal injection with 0.5 ml of a Ringer solution,in which 10 mg/kg of the peptide compound comprising 11R-VIVIT and FITCprepared in Example 1 was dissolved, as the injection. 6 hours afterinjection, the spleen, the lymph node, the liver, the kidney and theheart were removed from each mouse and each were frozen at −80° C. in 3ml of Histo Prep® (available from Fisher Scientific International,Inc.). Then, sections of 10 to 50 μm were prepared on a cryostat andanalyzed by a Zeiss microscope (made by Carl Zeiss, Inc.). As control,sections of an untreated C3H/HeN mouse were used.

As a result of analysis, in each of the sections of the spleen, thelymph node, the liver, the kidney and the heart derived from the mouseto which the complex peptide was administered, fluorescence of FITC wasobserved. On the other hand, in the control section, fluorescence ofFITC was not observed. This indicates that by intraperitonealadministration, 11R-VIVIT is introduced into at least the cells of thespleen, the lymph node, the liver, the kidney and the heart.

EXAMPLE 3

The dose dependant effect of 11R-VIVIT to transcription of IL-2 gene wasstudied.

Using 1 ml of 10% fetal bovine serum-containing RPMI (available fromInvitrogen BV) containing 1 nM, 10 nM, 100 nM or 1 μM of 11R-VIVIT,1×10⁵ Jurkat cells were cultured for 1 hour in a carbon dioxide culturedevice (made by Sanyo Denki Co., Ltd.) set to 37° C. and carbon dioxideconcentration of 5%. Subsequently, 100 μl of RPMI containing 2 μM ofphorbol 12-myristate 13-acetate (PMA) (available from Sigma-Aldrich Co.)and 40 μM of ionomycin (available from Sigma-Aldrich Co.) was added andculture was continued for 12 more hours. Then, the cells were recoveredand using the obtained cells, the transcription amount of IL-2 gene wasmeasured by the following real-time quantitative RT-PCR. In real-timequantitative RT-PCR, GAPDH, which is known as a housekeeping gene, wasused as the inner standard substance. Also, real-time quantitativeRT-PCR was conducted in the same manner, using Jurkat cells cultured ina medium without 11R-VIVIT as control cells.

RNeasy Mini Kit (available from QIAGEN K.K.) was used according toprotocol and total RNA was extracted from the cells. Using the obtainedtotal RNA, reverse transcription reaction was conducted by incubation at22° C. for 10 minutes and then at 42° C. for 20 minutes.

Thereafter, using Light Cycler-Fast Start DNA Master SYBR Green I Kit(available from Roche Molecular Biochemicals), 20 μl of a mixed reactionsolution (1/10 of the amount of the transcription product, 4 mM ofMgCl₂, 4 kinds of primers: 0.5 μM respectively, 2 μl of 10× LightCycler-Fast Start DNA Master SYBR Green I) was prepared foramplification reaction. As the primer for human IL-2, the primerincluded in Competitive Quantitative RT-PCR Kit (available fromFunakoshi Co., Ltd.) was used. As the primer for GAPDH,5′-CTGACCAGGGTCCTATTCCA-3′ (SEQ ID NO. 5) and 5′-TGGTTATCCCAAGCAAGAGG-3′(SEQ ID NO. 6) (both available from Gene Set) were used. Theamplification reaction was conducted by incubation at 95° C. for 10minutes and then 60 cycles of incubation at 94° C. for 15 seconds, 57°C. for 5 seconds and 72° C. for 10 seconds. Subsequently, thetemperature was raised to 95° C. at a rate of 0.2° C./second. Fordissociation curve analysis, Light Cycler Software (available from RocheMolecular Biochemicals) was used. The relative RNA amount to GAPDH foundby analysis is shown in FIG. 1 (n=3).

11R-VIVIT suppressed the transcription of IL-2 gene to 12% of the casewherein treatment was not conducted. IL-2 gene is a gene, in whichtranscription is promoted, when T cells are activated by allergies andrejection. Consequently, the results indicate that 11R-VIVIT cansuppress T cell activation.

COMPARATIVE EXAMPLE 1

A peptide compound comprising 11 consecutive arginine residues and VEETto which 3 glycine residues are added (hereinafter referred to as11R-VEET) was artificially synthesized and purified in the same manneras in Example 1. VEET is a peptide of the amino acid sequencerepresented by MAGPPHIVEETGPHVI (SEQ ID NO. 3). The peptide sequence of11R-VEET is shown in SEQ ID NO. 4. Hereinafter, 11R-VEET is used asnegative control of 11R-VIVIT.

Jurkat cells were cultured and RT-PCR was conducted using the obtainedcells in the same manner as in Example 3, except that a mediumcontaining 100 nM of FK 506 (available from Fujisawa Pharmaceutical Co.,Ltd.) or 1 μM of 11R-VEET, instead of 11R-VIVIT, was used. The resultsare shown in FIG. 1.

11R-VEET did not affect the transcription level of IL-2 gene.

EXAMPLE 4

The half-life of 11R-VIVIT in T cells was studied based on inhibitoryactivity to IL-2 transcription.

Using 1 ml of 10% fetal bovine serum-containing RPMI containing 1 μM of11R-VIVIT, 1×10⁵ Jurkat cells were cultured for 1 hour in a carbondioxide culture device set to 37° C. and carbon dioxide concentration of5%. Subsequently, 100 μl of RPMI containing 2 μM of PMA and 40 μM ofionomycin was added and culture was conducted further for 12 hours, 17hours, 23 hours, 35 hours or 59 hours. As control cells, Jurkat cells,cultured in the same manner as above for 1 hour in 10% fetal bovineserum-containing RPMI without 11R-VIVIT and then cultured further for 12hours, 17 hours, 23 hours, 35 hours or 59 hours after adding 100 μl ofRPMI containing 2 μM of PMA and 40 μM of ionomycin, were used.

Subsequently, the relative RNA amount to GAPDH was measured byconducting RT-PCR and real-time quantitative RT-PCR and analysis in thesame manner as in Example 3, except that the respective obtained cellswere used as samples. The results are shown in FIG. 2 (n=3). The timeshown in FIG. 2 refers to the total culture time.

The half-life of 11R-VIVIT was approximately 30 hours. 60 hours after11R-VIVIT was added, the IL-2 transcription inhibitory activity thereofcould not be observed.

COMPARATIVE EXAMPLE 2

Using 1 ml of 10% fetal bovine serum-containing RPMI containing 1 μM ofFK 506, 1×10⁵ Jurkat cells were cultured for 1 hour in a carbon dioxideculture device set to 37° C. and carbon dioxide concentration of 5%.Subsequently, 100 μl of RPMI containing 2 μM of PMA and 40 μM ofionomycin was added and culture was conducted further for 12 hours, 17hours, 23 hours, 35 hours or 59 hours. Thereafter, RT-PCR and real-timequantitative RT-PCR were conducted in the same manner as in Example 3,except that the obtained culture cells were used. The relative IL-2 mRNAlevel 60 hours after adding FK 506 is shown in FIG. 2.

FK 506 was not metabolized even after 60 hours and strongly suppressedtranscription of IL-2.

EXAMPLE 5

The effect of 11R-VIVIT to activation and proliferation of T cells wasstudied by mixed lymphocyte test.

Spleen cells were retrieved from a 6 to 8 week old C3H/HeN mouse and a 6to 8 week old BALB/c mouse H-2d (available from Shimizu LaboratorySupplies Co., Ltd.). The BALB/c spleen cells were treated with mitomycinC (available from Kyowa Hakko Kogyo Co., Ltd.) and used as stimulatingcells.

RPMI containing 1 pM, 1 nM or 1 μM of 11R-VIVIT was added to aflat-bottomed 96-well plate (available from Iwaki & Co., Ltd.).Subsequently, 5×10⁵ of mixed cells obtained by mixing the abovestimulating cells and the spleen cells derived from a C3H/HeN mouse in aratio of 1:5 were added in each well and cultured for 90 hours in acarbon dioxide culture device set to 37° C. and carbon dioxideconcentration of 5%. As control, the mixed cells were added to wellscontaining only a medium and cultured in the same manner.

Then, ³H-methyl-thymidine (hereinafter referred to as [³H]TdR, availablefrom Amersham Pharmacia Biotech K.K.) of 0.5 μCi/well was added andculture was conducted for 6 hours. Thereafter, the cells were placed onfilter paper having a diameter of 1 cm and the radioactivity of [³H]TdRincorporated into the cells was measured by a beta counter (made byBeckman Coulter K.K.). The results are shown in FIG. 3.

1 μM of 11R-VIVIT suppressed proliferation of lymphocytes by 43% basedon control. In FIG. 3, * indicates that P<0.05 in comparison to controland ** indicates that P<0.001.

COMPARATIVE EXAMPLE 3

Mixed cells were cultured, [3H]TdR was added and the radioactivity of[³H]TdR incorporated into the cells was measured by a beta counter inthe same manner as in Example 5, except that RPMI containing 1 pM, 1 nMor 1 μM of FK 506 or 1 μM of 11R-VEET instead of 11R-VIVIT was used. Theresults are shown in FIG. 3.

Proliferation of lymphocytes was not affected by 1 μM of 11R-VEET.

COMPARATIVE EXAMPLE 4

Cells were cultured, [³H]TdR was added and the radioactivity of [³H]TdRincorporated into the cells was measured by a beta counter in the samemanner as in Example 5, except that RPMI that does not contain 11R-VIVITwas used as the medium and only spleen cells derived from a C3H/HeNmouse was used instead of mixed cells. The results are shown in FIG. 3.

EXAMPLE 6

0.5 ml of a Ringer solution in which 10 mg/kg of 11R-VIVIT was dissolvedwas used as the injection and the effect of 11R-VIVIT to activation andproliferation of T cells in vivo was studied.

The above injection was injected intraperitoneally to a 6 week oldC3H/Hen mouse once a day for 2 days.

6 hours after the second injection, the spleen was surgically removed.The obtained spleen cells (1×10⁴ cells) were added to a flat-bottomed96-well plate together with spleen cells (1×10⁵ cells) of a BALB/c mousetreated with mitomycin C and cultured for 90 hours in a carbon dioxideculture device set to 37° C. and carbon dioxide concentration of 5%.RPMI was used as the medium. For control, the mixed cells were added towells containing only medium and cultured in the same manner.

Then, [³H]TdR of 0.5 μCi/well was added and culture was conducted for 6hours. Thereafter, the cells were placed on filter paper having adiameter of 1 cm and the radioactivity of [³H]TdR incorporated into thecells was measured by a beta counter. The results are shown in FIG. 4.In FIG. 4, ** indicates that P<0.001 in comparison to control.

10 mg/kg of 11R-VIVIT exhibited a suppression effect of 30% to controlin vivo.

COMPARATIVE EXAMPLE 5

A C3H/HeN mouse was subjected to intraperitoneal injection, spleen cellswere retrieved and cultured, [³H]TdR was added and the radioactivity of[³H]TdR incorporated into the cells was measured by a beta counter inthe same manner as in Example 6, except that 1 mg/kg of FK 506 or 10mg/kg of 11R-VEET was used, instead of 11R-VIVIT. The results are shownin FIG. 4.

Proliferation of lymphocytes was not affected by 1 μM of 11R-VEET.

COMPARATIVE EXAMPLE 6

Cells were cultured, [³H]TdR was added and the radioactivity of [³H]TdRincorporated into the cells was measured by a beta counter in the samemanner as in Example 6, except that RPMI without 11R-VIVIT was used asthe medium and spleen cells derived from a C3H/HeN mouse were usedinstead of mixed cell. The results are shown in FIG. 4.

EXAMPLE 7

0.5 ml of a Ringer solution in which 10 mg/kg of 11R-VIVIT was dissolvedwas used as the injection and the effect of 11R-VIVIT in beta celltransplant was studied. For the transplant, a 6 week old BALB/c mousewas used as the donor and a 6 week old C3H/HeN mouse was used as therecipient.

A diabetic model mouse was prepared by intraperitoneally injecting 220mg/kg of streptozocin (available from Sigma-Aldrich Japan K.K.) to aC3H/HeN mouse. 6 days after the streptozocin injection, the mouse inwhich the glucose level exceeded 350 mg/dl was determined as beinghyperglycemic.

Meanwhile, the spleen of the BALB/c mouse was taken out and aftersubjecting to discontinuous Ficoll density gradient centrifugation, betacells were isolated by normal collagenase digestion.

About 500 of the isolated beta cells were transplanted under thecapusula of the left kidney of the above diabetic model mouse. After thetransplant, 10 mg/kg of 11R-VIVIT was intraperitoneally injected to themouse once a day (n=6). For control, physiologic saline that does notcontain 11R-VIVIT was injected in the mouse to which the beta cells weretransplanted (n=4). The glycemia was measured and when the level thereofexceeded 200 mg/dl for 2 consecutive days, transplant rejection wasconsidered to have occurred. The results evaluated by Kaplan-Meier areshown in FIG. 5.

By administering 11R-VIVIT, the survival rate increased significantlycompared to control (P<0.005).

COMPARATIVE EXAMPLE 7

The effect of 11R-VEET to beta cell transplant was studied in the samemanner as in Example 7, except that 10 mg/kg of 11R-VEET was usedinstead of 11R-VIVIT. The results are shown in FIG. 5.

As in the case of control, 11R-VEET did not affect the survival of thetransplant.

EXAMPLE 8

Using the mouse to which 11R-VIVIT was administered in Example 1, thefunction of the transplanted beta cells were studied.

50 days after the beta cell transplant, the kidney was surgicallyremoved. Then, using frozen mitochrome (available from Carl Zeiss,Inc.), a 30 μm kidney section was prepared. The obtained section wassubjected to immunostaining using a polyclonal antibody against insulin(available from Santa Cruz Biotechnology, Inc.), avidin and biotinperoxidase method (available from Vector Laboratories, Inc.).

The results of immunostaining are shown in FIG. 6. In FIG. 6, 1represents the transplanted beta cells and 2 represents the kidney ofthe recipient. The transplanted cells were confirmed to produce insulin,as the transplanted beta cells were stained by the anti-insulinantibody.

EXAMPLE 9

The effect of 11R-VIVIT to insulin secretion of βTC6 cells was studied.βTC6 cells are cell lines of islets of Langerhans, which secrete insulinby glucose response.

βTC6 cells (available from American Type Culture Collection, CRL-11506)were added to a 96 well plate (5×10⁴ cell/well) and cultured for 96hours using 1 ml of 10% fetal bovine serum-containing RPMI containing10, 100, 1000 or 10000 nM of 11R-VIVIT in a carbon dioxide culturedevice set to 37° C. and carbon dioxide concentration of 5%. The mediumwas replaced with a fresh medium containing 11R-VIVIT every 24 hours.After 96 hours, the medium was replaced with a fresh medium. Culture wasconducted for 1 more hour and the culture supernatant was collected. Forcontrol, βTC6 cells were cultured in the same manner, except that amedium without 11R-VIVIT was used.

Subsequently, using the collected supernatant, the secreted insulin wasanalyzed using Mouse Insulin ELISA (TMB) Kit (available from ShibayagiCo., Ltd.) according to protocol. FIG. 7(a) shows the amount of insulinsecretion of cells treated with 11R-VIVIT by a relative value tocontrol.

The insulin secretion amount of βTC6 cells was almost the same ascontrol when the amount of 11R-VIVIT was within the range of 0 to 1 μM.However, when the amount of 11R-VIVIT was 10 μM, the insulin secretionamount was 71% of control (P=0.042).

COMPARATIVE EXAMPLE 8

βTC6 cells were cultured and the effect to insulin secretion of βTC6cells was studied in the same manner as in Example 9, except that acomplete medium containing 1, 10, 100 or 1000 nM of FK 506 or 10, 100,1000 or 10000 nM of 11R-VEET, instead of 11R-VIVIT, was used. Theresults are shown in FIGS. 7(b) and 7(c).

The insulin secretion amount of βTC6 cells was significantly decreasedby FK 506 and unaffected by 11R-VEET.

EXAMPLE 10

The effect of 11R-VIVIT to proliferation of βTC6 cells was studied.

βTC6 cells were added to a 96 well plate (5×10⁴ cell/well) and culturedfor 88 hours using a complete medium containing 10, 100, 1000 or 10000nM of 11R-VIVIT in a carbon dioxide culture device set to 37° C. andcarbon dioxide concentration of 5%. The medium was replaced with a freshmedium containing 11R-VIVIT every 24 hours. Subsequently, [³H]TdR of 1μCi/well was added and culture was conducted for 8 more hours. Forcontrol, βTC6 cells were cultured in the same manner except that amedium without 11R-VIVIT was used. Thereafter, the cells were placed onfilter paper having a diameter of 1 cm and the radioactivity of [³H]TdRincorporated into the cells was measured by a beta counter. FIG. 8(a)shows proliferation of cells treated with 11R-VIVIT by a relative valueto control.

Proliferation of βTC6 cells was almost the same as control when theamount of 11R-VIVIT was within the range of 0 to 1 μM. When the amountof 11R-VIVIT was 10 μM, proliferation was 73% of control (P=0.075).

COMPARATIVE EXAMPLE 9

βTC6 cells were cultured and the effect to proliferation of βTC6 cellswas studied in the same manner as in Example 10, except that a completemedium containing 1, 10, 100 or 1000 nM of FK 506 or 10, 100, 1000 or10000 nM of 11R-VEET, instead of 11R-VIVIT, was used. The results areshown in FIGS. 8(b) and 8(c).

FK 506 and 11R-VEET did not exhibit an inhibitory effect toproliferation of βTC6 cells.

EXAMPLE 11

The effect of 11R-VIVIT to cardiac hypertrophy was studied.

A cardiac hypertrophy model rat was hypodermically injected every otherday with 0.5 ml of Ringer solution in which 1.5 mg/kg of 11R-VIVIT wasdissolved (n=6). The cardiac hypertrophy model rat was prepared byligating the aorta of a 7 week old male Wistar rat having weight of 200to 250 g (available from Shimizu Laboratory Supplies Co., Ltd.) andapplying pressure load. In the present specification, the cardiachypertrophy model rat is referred to as AoB.

Subsequently, the heart of the rat was evaluated by the followingmethods after 0, 1, 2, 3, 4, and 5 weeks. As control, a normal rat towhich 11R-VIVIT was not administered was used.

Control and 11R-VIVIT administered AoB were subjected to ultrasonic echotest (ULC) and preparation of tissue specimen and blood test wereconducted in the following manner.

ULC was conducted by analyzing the thickness of the interventricularseptum and left ventricle anterior wall of the rat by ultrasonic echo(made by Hitachi, Ltd.) using a pediatric probe. The results are shownin FIGS. 9(a) and 9(b). As a result, the interventricular septum wallthickness and the left ventricle anterior wall thickness of AoB wereboth found to improve by administration of 11R-VIVIT.

The tissue specimen was prepared by surgically removing the rat heart,preparing a section of 5 μm thickness by frozen microtome and stainingthe obtained section by hematoxylin-eosin staining. The obtained tissuespecimen was observed with a microscope (FIG. 10(d)). In FIG. 10, 3represents the left ventricle and 4 represents the right ventricle. As aresult, the symptoms were found to be improved to conditions close tonormal by administration of 11R-VIVIT.

Measurement of atrial natriuretic peptide (ANP) and B-type natriureticpeptide (BNP) was commissioned to SRL, Inc. The results are shown inFIGS. 11(a) and 11(b). As a result, the value of ANP and BNP in AoB werefound to improve by administration of 11R-VIVIT.

COMPARATIVE EXAMPLE 10

Measurement of ULC, preparation of tissue specimen and blood test wasconducted in the same manner as in Example 11, except that AoB to which11R-VIVIT was not administered was used. The results are shown in FIGS.9(a), 9(b), 10(b), 11(a) and 11(b).

COMPARATIVE EXAMPLE 11

The effect of CysA to cardiac hypertrophy was studied in the same manneras in Example 11, except that 5 mg/kg of CysA was used instead of11R-VIVIT. The results are shown in FIGS. 9(a), 9(b), 10(c), 11(a) and11(b).

TEST EXAMPLE 1

The toxicity of 11R-VIVIT to hepatic function and renal function wasstudied using 11R-VIVIT administered AoB.

AoB was hypodermically injected every other day for 4 weeks with 0.5 mlof Ringer solution in which 1.5 mg/kg of 11R-VIVIT was dissolved (n=6).Measurement of the following for the obtained AoB was commissioned toSRL, Inc.

As the hepatic function test, glutamate oxaloacetate transaminase (GOT)and glutamate pyruvate transaminase (GPT) were measured and as the renalfunction test, creatinine (Cr) and urea nitrogen (BUN) were measured. Anormal rat and AoB to which 11R-VIVIT was not administered were used ascontrol. The results are shown in Table 1. TABLE 1 11R-VIVITadministered Control AoB AoB GOT 81.2 ± 4.35 94.2 ± 14.9 99.2 ± 6.24(IU/l) GPT 49.6 ± 2.42 63.1 ± 14.6 55.8 ± 9.95 (IU/l) BUN 16.3 ± 1.1321.5 ± 1.38 16.6 ± 2.71 (mg/dl) Cr 0.27 ± 0.02 0.37 ± 0.03 0.28 ± 0.02(mg/dl)

The measurement results suggest that the peptide compound of the presentinvention does not affect the functions of the liver and kidney.

INDUSTRIAL APPLICABILITY

The peptide compound of the present invention can be used as atherapeutic agent for diseases caused by activation of NFAT havingextremely few side effects. Specifically, the peptide compound isextremely useful, as the compound can be used for example, as animmunosuppressive agent and a cardiac hypertrophy suppressive agent.

SEQUENCE LISTING FREE TEXT

-   SEQ ID NO. 2: fused peptide sequence-   SEQ ID NO. 4: fused peptide sequence

1. A peptide compound comprising 9 to 13 residues of arginine and theamino acid sequence of SEQ ID NO.
 1. 2. The peptide compound of claim 1,comprising 11 residues of arginine.
 3. A NFAT activation inhibitoryagent comprising the peptide compound of claim
 1. 4. Animmunosuppressive agent comprising the peptide compound of claim 1 as anactive ingredient.
 5. A cardiac hypertrophy suppressive agent comprisingthe peptide compound of claim 1 as an active ingredient.
 6. A NFATactivation inhibitory agent comprising the peptide compound of claim 2.7. An immunosuppressive agent comprising the peptide compound of claim 2as an active ingredient.
 8. A cardiac hypertrophy suppressive agentcomprising the peptide compound of claim 2 as an active ingredient.